Skip to main content
Genetics logoLink to Genetics
. 1993 Aug;134(4):1039–1044. doi: 10.1093/genetics/134.4.1039

Antimutator Mutations in the α Subunit of Escherichia Coli DNA Polymerase III: Identification of the Responsible Mutations and Alignment with Other DNA Polymerases

I J Fijalkowska 1, R M Schaaper 1
PMCID: PMC1205572  PMID: 8375647

Abstract

The dnaE gene of Escherichia coli encodes the DNA polymerase (α subunit) of the main replicative enzyme, DNA polymerase III holoenzyme. We have previously identified this gene as the site of a series of seven antimutator mutations that specifically decrease the level of DNA replication errors. Here we report the nucleotide sequence changes in each of the different antimutator dnaE alleles. For each a single, but different, amino acid substitution was found among the 1,160 amino acids of the protein. The observed substitutions are generally nonconservative. All affected residues are located in the central one-third of the protein. Some insight into the function of the regions of polymerase III containing the affected residues was obtained by amino acid alignment with other DNA polymerases. We followed the principles developed in 1990 by M. Delarue et al. who have identified in DNA polymerases from a large number of prokaryotic and eukaryotic sources three highly conserved sequence motifs, which are suggested to contain components of the polymerase active site. We succeeded in finding these three conserved motifs in polymerase III as well. However, none of the amino acid substitutions responsible for the antimutator phenotype occurred at these sites. This and other observations suggest that the effect of these mutations may be exerted indirectly through effects on polymerase conformation and/or DNA/polymerase interactions.

Full Text

The Full Text of this article is available as a PDF (1.7 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bebenek K., Joyce C. M., Fitzgerald M. P., Kunkel T. A. The fidelity of DNA synthesis catalyzed by derivatives of Escherichia coli DNA polymerase I. J Biol Chem. 1990 Aug 15;265(23):13878–13887. [PubMed] [Google Scholar]
  2. Blanco L., Bernad A., Blasco M. A., Salas M. A general structure for DNA-dependent DNA polymerases. Gene. 1991 Apr;100:27–38. doi: 10.1016/0378-1119(91)90346-d. [DOI] [PubMed] [Google Scholar]
  3. Blanco L., Bernad A., Salas M. Evidence favouring the hypothesis of a conserved 3'-5' exonuclease active site in DNA-dependent DNA polymerases. Gene. 1992 Mar 1;112(1):139–144. doi: 10.1016/0378-1119(92)90316-h. [DOI] [PubMed] [Google Scholar]
  4. Brenowitz S., Kwack S., Goodman M. F., O'Donnell M., Echols H. Specificity and enzymatic mechanism of the editing exonuclease of Escherichia coli DNA polymerase III. J Biol Chem. 1991 Apr 25;266(12):7888–7892. [PubMed] [Google Scholar]
  5. Delarue M., Poch O., Tordo N., Moras D., Argos P. An attempt to unify the structure of polymerases. Protein Eng. 1990 May;3(6):461–467. doi: 10.1093/protein/3.6.461. [DOI] [PubMed] [Google Scholar]
  6. Fijalkowska I. J., Dunn R. L., Schaaper R. M. Mutants of Escherichia coli with increased fidelity of DNA replication. Genetics. 1993 Aug;134(4):1023–1030. doi: 10.1093/genetics/134.4.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gillin F. D., Nossal N. G. Control of mutation frequency by bacteriophage T4 DNA polymerase. I. The CB120 antimutator DNA polymerase is defective in strand displacement. J Biol Chem. 1976 Sep 10;251(17):5219–5224. [PubMed] [Google Scholar]
  8. Goodman M. F. DNA replication fidelity: kinetics and thermodynamics. Mutat Res. 1988 Jul-Aug;200(1-2):11–20. doi: 10.1016/0027-5107(88)90067-x. [DOI] [PubMed] [Google Scholar]
  9. Ito J., Braithwaite D. K. Compilation and alignment of DNA polymerase sequences. Nucleic Acids Res. 1991 Aug 11;19(15):4045–4057. doi: 10.1093/nar/19.15.4045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jung G. H., Leavitt M. C., Hsieh J. C., Ito J. Bacteriophage PRD1 DNA polymerase: evolution of DNA polymerases. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8287–8291. doi: 10.1073/pnas.84.23.8287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kohlstaedt L. A., Wang J., Friedman J. M., Rice P. A., Steitz T. A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. doi: 10.1126/science.1377403. [DOI] [PubMed] [Google Scholar]
  12. Kraft R., Tardiff J., Krauter K. S., Leinwand L. A. Using mini-prep plasmid DNA for sequencing double stranded templates with Sequenase. Biotechniques. 1988 Jun;6(6):544-6, 549. [PubMed] [Google Scholar]
  13. Kuchta R. D., Benkovic P., Benkovic S. J. Kinetic mechanism whereby DNA polymerase I (Klenow) replicates DNA with high fidelity. Biochemistry. 1988 Sep 6;27(18):6716–6725. doi: 10.1021/bi00418a012. [DOI] [PubMed] [Google Scholar]
  14. Maki H., Kornberg A. Proofreading by DNA polymerase III of Escherichia coli depends on cooperative interaction of the polymerase and exonuclease subunits. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4389–4392. doi: 10.1073/pnas.84.13.4389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Maki H., Mo J. Y., Sekiguchi M. A strong mutator effect caused by an amino acid change in the alpha subunit of DNA polymerase III of Escherichia coli. J Biol Chem. 1991 Mar 15;266(8):5055–5061. [PubMed] [Google Scholar]
  16. Polesky A. H., Dahlberg M. E., Benkovic S. J., Grindley N. D., Joyce C. M. Side chains involved in catalysis of the polymerase reaction of DNA polymerase I from Escherichia coli. J Biol Chem. 1992 Apr 25;267(12):8417–8428. [PubMed] [Google Scholar]
  17. Polesky A. H., Steitz T. A., Grindley N. D., Joyce C. M. Identification of residues critical for the polymerase activity of the Klenow fragment of DNA polymerase I from Escherichia coli. J Biol Chem. 1990 Aug 25;265(24):14579–14591. [PubMed] [Google Scholar]
  18. Schaaper R. M. The mutational specificity of two Escherichia coli dnaE antimutator alleles as determined from lacI mutation spectra. Genetics. 1993 Aug;134(4):1031–1038. doi: 10.1093/genetics/134.4.1031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Scheuermann R. H., Echols H. A separate editing exonuclease for DNA replication: the epsilon subunit of Escherichia coli DNA polymerase III holoenzyme. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7747–7751. doi: 10.1073/pnas.81.24.7747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Studwell P. S., O'Donnell M. Processive replication is contingent on the exonuclease subunit of DNA polymerase III holoenzyme. J Biol Chem. 1990 Jan 15;265(2):1171–1178. [PubMed] [Google Scholar]
  21. Takeshita S., Sato M., Toba M., Masahashi W., Hashimoto-Gotoh T. High-copy-number and low-copy-number plasmid vectors for lacZ alpha-complementation and chloramphenicol- or kanamycin-resistance selection. Gene. 1987;61(1):63–74. doi: 10.1016/0378-1119(87)90365-9. [DOI] [PubMed] [Google Scholar]
  22. Tomasiewicz H. G., McHenry C. S. Sequence analysis of the Escherichia coli dnaE gene. J Bacteriol. 1987 Dec;169(12):5735–5744. doi: 10.1128/jb.169.12.5735-5744.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wang T. S., Wong S. W., Korn D. Human DNA polymerase alpha: predicted functional domains and relationships with viral DNA polymerases. FASEB J. 1989 Jan;3(1):14–21. doi: 10.1096/fasebj.3.1.2642867. [DOI] [PubMed] [Google Scholar]
  24. Welch M. M., McHenry C. S. Cloning and identification of the product of the dnaE gene of Escherichia coli. J Bacteriol. 1982 Oct;152(1):351–356. doi: 10.1128/jb.152.1.351-356.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES